Showing papers on "Oxoglutarate dehydrogenase complex published in 2016"

The pyruvate dehydrogenase complex in cancer: An old metabolic gatekeeper regulated by new pathways and pharmacological agents.

Abstract: Cancer cells exhibit an altered metabolism which is characterized by a preference for aerobic glycolysis more than mitochondrial oxidation of pyruvate. This provides anabolic support and selective growth advantage for cancer cells. Recently, a new concept has arisen suggesting that these metabolic changes may be due, in part, to an attenuated mitochondrial function which results from the inhibition of the pyruvate dehydrogenase complex (PDC). This mitochondrial complex links glycolysis to the Krebs cycle and the current understanding of its regulation involves the cyclic phosphorylation and dephosphorylation by specific pyruvate dehydrogenase kinases (PDKs) and pyruvate dehydrogenase phosphatases (PDPs).

A contemporary perspective on the molecular characteristics of mitochondrial autoantigens and diagnosis in primary biliary cholangitis.

Abstract: Primary biliary cholangitis (PBC) is an autoimmune hepatobiliary disease characterized by immune mediated destruction of the intrahepatic small bile ducts and the presence of antimitochondrial antibodies (AMAs). The mitochondrial autoantigens have been identified as the E2 subunits of the 2-oxo-acid dehydrogenase complex, including the E2 subunits of pyruvate dehydrogenase, branched-chain 2-oxo acid dehydrogenase complex, oxoglutarate dehydrogenase complex, E3 binding protein and PDC E1 alpha subunit. The AMA epitope is mapped within the E2 lipoic acid binding domain, which is particularly important for oxidative phosphorylation. In addition, lipoic acid, which serves as a swinging arm to capture electrons, is particularly susceptible to an electrophilic attack and may provide clues to the etiology of PBC. This review emphasizes the molecular characteristics of AMAs, including detection, immunochemistry and the putative role in disease. These data have significance not only specifically for PBC, but generically for autoimmunity.

Rearrangement of Mitochondrial Pyruvate Dehydrogenase Subunit Dihydrolipoamide Dehydrogenase Protein‐Protein Interactions by the MDM2 Ligand Nutlin‐3

Abstract: Drugs targeting MDM2's hydrophobic pocket activate p53. However, these agents act allosterically and have agonist effects on MDM2's protein interaction landscape. Dominant p53-independent MDM2-drug responsive-binding proteins have not been stratified. We used as a variable the differential expression of MDM2 protein as a function of cell density to identify Nutlin-3 responsive MDM2-binding proteins that are perturbed independent of cell density using SWATH-MS. Dihydrolipoamide dehydrogenase, the E3 subunit of the mitochondrial pyruvate dehydrogenase complex, was one of two Nutlin-3 perturbed proteins identified fours hour posttreatment at two cell densities. Immunoblotting confirmed that dihydrolipoamide dehydrogenase was induced by Nutlin-3. Depletion of MDM2 using siRNA also elevated dihydrolipoamide dehydrogenase in Nutlin-3 treated cells. Mitotracker confirmed that Nutlin-3 inhibits mitochondrial activity. Enrichment of mitochondria using TOM22+ immunobeads and TMT labeling defined key changes in the mitochondrial proteome after Nutlin-3 treatment. Proximity ligation identified rearrangements of cellular protein–protein complexes in situ. In response to Nutlin-3, a reduction of dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase protein complexes highlighted a disruption of the pyruvate dehydrogenase complex. This coincides with an increase in MDM2/dihydrolipoamide dehydrogenase complexes in the nucleus that was further enhanced by the nuclear export inhibitor Leptomycin B. The data suggest one therapeutic impact of MDM2 drugs might be on the early perturbation of specific protein–protein interactions within the mitochondria. This methodology forms a blueprint for biomarker discovery that can identify rearrangements of MDM2 protein–protein complexes in drug-treated cells.

Mitochondrial Carriers Link the Catabolism of Hydroxyaromatic Compounds to the Central Metabolism in Candida parapsilosis

Abstract: The pathogenic yeast Candida parapsilosis metabolizes hydroxyderivatives of benzene and benzoic acid to compounds channeled into central metabolism, including the mitochondrially localized tricarboxylic acid cycle, via the 3-oxoadipate and gentisate pathways. The orchestration of both catabolic pathways with mitochondrial metabolism as well as their evolutionary origin is not fully understood. Our results show that the enzymes involved in these two pathways operate in the cytoplasm with the exception of the mitochondrially targeted 3-oxoadipate CoA-transferase (Osc1p) and 3-oxoadipyl-CoA thiolase (Oct1p) catalyzing the last two reactions of the 3-oxoadipate pathway. The cellular localization of the enzymes indicates that degradation of hydroxyaromatic compounds requires a shuttling of intermediates, cofactors, and products of the corresponding biochemical reactions between cytosol and mitochondria. Indeed, we found that yeast cells assimilating hydroxybenzoates increase the expression of genes SFC1, LEU5, YHM2, and MPC1 coding for succinate/fumarate carrier, coenzyme A carrier, oxoglutarate/citrate carrier, and the subunit of pyruvate carrier, respectively. A phylogenetic analysis uncovered distinct evolutionary trajectories for sparsely distributed gene clusters coding for enzymes of both pathways. Whereas the 3-oxoadipate pathway appears to have evolved by vertical descent combined with multiple losses, the gentisate pathway shows a striking pattern suggestive of horizontal gene transfer to the evolutionarily distant Mucorales.

Method for producing alpha-oxoglutarate under catalysis of L-glutamate oxidase

Abstract: The invention discloses a method for producing alpha-oxoglutarate under catalysis of L-glutamate oxidase. The method comprises the following steps: performing optimal design and synthesis on codon of an L-glutamate oxidase (L-GOX) gene; constructing a fusion expression vector pET-28a-LGOX-ELP of the L-GOX and Elastin-Like Proteins (ELP), transferring the vector into escherichia coli Rosetta (DE3), selecting a converter for cultivation, adding an inducer for expression, and obtaining recombined L-glutamate oxidase fusion protein crude enzyme fluid; performing purification by an ITC method, thus obtaining recombined L-glutamate oxidase fusion proteins, performing enzyme digestion on the recombined L-glutamate oxidase fusion proteins by protease, and obtaining mature L-glutamate oxidase; producing oxoglutarate under the catalysis of the mature L-glutamate oxidase. The method is simple, quick and efficient, can be used for large-scale separation and purification of L-glutamate oxidase, and is higher in actual application value in the enzyme production of oxoglutarate.

Activity of liver mitochondrial Krebs cycle NAD+-dependent dehydrogenases in rats with hepatitis induced by acetaminophen under conditions of alimentary protein deficiency

Abstract: Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD+/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deficiency. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD+/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein deficiency caused a more pronounced decrease in the activity of studied Krebs cycle NAD+-dependent dehydrogenases and a 2.2-fold increase of the mitochondrial NAD+/NADН ratio.

[High efficient co-expression of leucine dehydrogenase and glucose dehydrogenase in Escherichia coli].

Abstract: Objective Different co-expression strategies to express leucine dehydrogenase and glucose dehydrogenase in E. coli were done to observe the effect of expression of different enzyme. A recombinant strain with two high enzyme activities was built for efficiently asymmetric synthesis of L-tert-leucine. Methods The leucine dehydrogenase (ldh) from Bacillus cereus and glucose dehydrogenase (gdh) from Bacillus sp. were co-expressed by three different strategies, including co-expressing two genes in single vector, co-expressing two genes in two vectors and expressing fusion protein. The catalytic efficiencies of recombinant strains with different enzyme activity ratio in different modes of biocatalyst were compared to produce L-tert-leucine from its corresponding α-keto acids. Results Different co-expression strategies displayed a slight impact on leucine dehydrogenase expression, whereas, a greater impact on glucose dehydrogenase. All the activity of leucine dehydrogenase was normally expressed, but the fusion proteins lost the activity of glucose dehydrogenase. Besides, the activity of glucose dehydrogenase was also totally inhibited when the 6-histidine tag was fused at C termini, which indicated the additional 6-histidine tag considerately depressed the glucose dehydrogenase activity. After optimization of expression, three recombinant strains exhibiting high enzyme activity and different enzyme activity ratio were used to synthesis L-tert-leucine in the mode of cell-free extracts and whole-cell. Result displayed a great influence on the catalytic efficiencies resulted from the mode of catalyst and enzyme activity. When the cell-free crude culture broth of E. coli BL21/pET28a-L-SD-AS-G coexpressing two genes in single vector was used as biocatalyst, 15 g/L cell loading and 0.1 mmol/L NAD+ were enough to completely transform 0.5 mol/L trimethylpyruvate into L-tert-leucine. Conclusion The recombinant strain with high activities of leucine dehydrogenase and glucose dehydrogenase was achieved by co-expressing two genes in single vector without histidine tag in E. coli and L-tert-leucine was efficiently produced with this recombinant strain.

Enzyme activity detection system and method of Fe and alpha-oxoglutarate dependence dioxygenases

Abstract: The invention provides an enzyme activity detection method of Fe and alpha-oxoglutarate dependence dioxygenases, comprising a step of detecting the enzyme activity of the enzyme by a color reaction of inorganic phosphate generated by a coupled reaction of Fe and oxoglutarate dependence dioxygenases and succinyl-coenzyme A synthetase and ammonium molybdate malachite green dye. In addition, the invention also provides an enzyme activity detection system of dioxygenase, a kit and application of thereof.